Introduction to the Theory and Practice of Fixation of Tissues

Eltoum, Isam A.; Fredenburgh, Jerry; Myers, Richard S.; Grizzle, William E.

doi:10.1179/his.2001.24.3.173

Cited by 169 publications

(111 citation statements)

References 29 publications

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“…49 Ethanol fixes tissue by altering the solubility of dissolved proteins, causing them to precipitate and agglomerate; however, this is an unlikely contributor since such fixation requires >50% ethanol. 50 After evaluating compaction in isolation, we incorporated it into a crosslinking treatment for the ABNP scaffolds. Compaction and crosslinking significantly increased the DMA properties of the ABNP, was comparable to the bNP control, and tended to be marginally higher than other crosslinked groups (Figure 4).…”

Section: Discussionmentioning

confidence: 99%

Ethanol-Mediated Compaction and Crosslinking Enhance Mechanical Properties and Degradation Resistance While Maintaining Cytocompatibility of a Nucleus Pulposus Scaffold

Walters

Gill

Mercuri

2018

Preprint

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Intervertebral disc degeneration is a complex, cell-mediated process originating in the nucleus pulposus (NP) and is associated with extracellular matrix catabolism leading to disc height loss and impaired spine kinematics. Previously, we developed an acellular bovine NP (ABNP) for NP replacement that emulated human NP matrix composition and supported cell seeding; however, its mechanical properties were lower than those reported for human NP. To address this, we investigated ethanol-mediated compaction and crosslinking to enhance the ABNP's dynamic mechanical properties and degradation resistance while maintaining its cytocompatibility. First, volumetric and mechanical effects of compaction only were confirmed by evaluating scaffolds after various immersion times in buffered 28% ethanol. It was found that compaction reached equilibrium at ~30% compaction after 45 min, and dynamic mechanical properties significantly increased 2-6x after 120 min of submersion. This was incorporated into a crosslinking treatment, through which scaffolds were subjected to 120 min pre-compaction in buffered 28% ethanol prior to carbodiimide crosslinking. Their dynamic mechanical properties were evaluated before and after accelerated degradation by ADAMTS-5 or MMP-13. Cytocompatibility was determined by seeding stem cells onto scaffolds and evaluating viability through metabolic activity and fluorescent staining. Compacted and crosslinked scaffolds showed significant increases in DMA properties without detrimentally altering their cytocompatibility, and these mechanical gains were maintained following enzymatic exposure.Low back pain (LBP) is the leading cause of Years Lived with Disability in all developed countries, 1 with a lifetime prevalence of 70-84%. 2 LBP has severe economic ramifications on the individual and societal levels, leading to premature workforce departure, short/long-term fiscal losses, 3 and cumulative yearly costs exceeding $100 billion in the US. 4 The most common diagnosis for patients experiencing LBP is intervertebral disc (IVD) degeneration (IDD). 5IVDs are avascular, fibrocartilaginous structures connecting adjacent vertebrae in the spinal column. Each IVD comprises an aggrecan-rich core known as the nucleus pulposus (NP), 15-25 concentric sheets of aligned collagen type I encircling the NP known as the annulus fibrosus (AF), and thin layers of hyaline cartilage covering the cranial and caudal surfaces of the IVD known as the cartilage endplates (CEP). 6 Adjacent vertebrae interface with the IVD through the CEP, which sequesters the NP within the disc while allowing for nutrient exchange with vertebral blood vessels. 7 The encapsulation of the NP and its anionic, dense matrix impart triphasic timedependence to its mechanical properties, allowing it to dissipate loads via fluid exudation and prevent harmful stress concentrations in AF and CEP through hydraulic pressurization. 6,8,9 IDD is a multi-factorial, cell-mediated process originating in the NP in which normal tissue turnover becomes imbalanced, favori...

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Section: Discussionmentioning

confidence: 99%

Ethanol-Mediated Compaction and Crosslinking Enhance Mechanical Properties and Degradation Resistance While Maintaining Cytocompatibility of a Nucleus Pulposus Scaffold

Walters

Gill

Mercuri

2018

Preprint

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“…However, the fixation techniques employed in this study, which were modified from Peng et al (2011), disrupted the internal architecture of the majority of cells in such a way that internal organelles could not be readily recognised. The reasons for this are believed to be twofold: first, coagulant fixatives such as TCA are known to result in cytoplasmic flocculation (Eltoum et al, 2001) and second, dehydration of cells results in a loss of hydrogen bonding that may cause bonding between molecules that would not normally interact, resulting in the fusion of cellular organelles (Wolkers et al, 2002). Recent research has since shown that these often considered pervasive fixation artefacts can be investigated, monitored and minimised using correlative complementary IR spectroscopy and single molecule localisation microscopy techniques (Whelan & Bell, 2015).…”

Section: Discussionmentioning

confidence: 99%

Chemical imaging of a Symbiodinium sp. cell using synchrotron infrared microspectroscopy: a feasibility study

Gordon

Martin

Bambery

et al. 2017

Journal of Microscopy

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The symbiotic relationship between corals and Symbiodinium spp. is the key to the success and survival of coral reef ecosystems the world over. Nutrient exchange and chemical communication between the two partners provides the foundation of this key relationship, yet we are far from a complete understanding of these processes. This is due, in part, to the difficulties associated with studying an intracellular symbiosis at the small spatial scales required to elucidate metabolic interactions between the two partners. This feasibility study, which accompanied a more extensive investigation of fixed Symbiodinium cells (data unpublished), examines the potential of using synchrotron radiation infrared microspectroscopy (SR-IRM) for exploring metabolite localisation within a single Symbiodinium cell. In doing so, three chemically distinct subcellular regions of a single Symbiodinium cell were established and correlated to cellular function based on assignment of diagnostic chemical classes.

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“…The most common fixatives are 4% formaldehyde or paraformaldehyde (PFA) in phosphate buffered saline (PBS; Nakamura, Nakamura & Hamada, 2013;Neufeld et al, 2013;Kernohan & Bérubé, 2014;Shiura et al, 2014;Oka & Sato, 2015;Thiruketheeswaran, Kiehl & D'Haese, 2016). Formaldehyde is a crosslinking fixative that forms covalent links between macromolecules such as lipids, peptides and DNA; this creates a mesh inside the cells or tissues to hold their components in place and minimize enzymatic degradation over time (Eltoum et al, 2001). PFA solutions produced from a powder will contain pure fixative, however, prepared 4% PFA solutions will produce polymers over time and become less effective as the polymers precipitate from the solution (Thavarajah et al, 2012).…”

Section: Tissue Preparation and Permeabilizationmentioning

confidence: 99%

“…As an alternative to formaldehyde, some protocols employ alcohol-based fixation using either ethanol (Schurter, LeBrun & Harrison, 2002) or methanol (Legendre et al, 2013). Ethanol and methanol are coagulant fixatives that replace free water in the tissue to dehydrate cells and destabilize hydrophobic and hydrogen bonds (Eltoum et al, 2001). Alcohol-based fixation is common for cultured cells and ice-cold (−20 C) ethanol and methanol have been used to fix multiple cultured cell lines in as little as 10 min (Shaffer et al, 2013).…”

Section: Tissue Preparation and Permeabilizationmentioning

confidence: 99%

A technical review and guide to RNA fluorescence in situ hybridization

Young

Jackson

Wyeth

2020

PeerJ

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RNA-fluorescence in situ hybridization (FISH) is a powerful tool to visualize target messenger RNA transcripts in cultured cells, tissue sections or whole-mount preparations. As the technique has been developed over time, an ever-increasing number of divergent protocols have been published. There is now a broad selection of options available to facilitate proper tissue preparation, hybridization, and post-hybridization background removal to achieve optimal results. Here we review the technical aspects of RNA-FISH, examining the most common methods associated with different sample types including cytological preparations and whole-mounts. We discuss the application of commonly used reagents for tissue preparation, hybridization, and post-hybridization washing and provide explanations of the functional roles for each reagent. We also discuss the available probe types and necessary controls to accurately visualize gene expression. Finally, we review the most recent advances in FISH technology that facilitate both highly multiplexed experiments and signal amplification for individual targets. Taken together, this information will guide the methods development process for investigators that seek to perform FISH in organisms that lack documented or optimized protocols. Seviour RJ. 2005. Improved permeabilization protocols for fluorescence in situ hybridization (FISH) of mycolic-acid-containing bacteria found in foams.Kislauskis EH, Li Z, Singer RH, Taneja KL. 1993. Isoform-specific 3′-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments.

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Introduction to the Theory and Practice of Fixation of Tissues

Cited by 169 publications

References 29 publications

Ethanol-Mediated Compaction and Crosslinking Enhance Mechanical Properties and Degradation Resistance While Maintaining Cytocompatibility of a Nucleus Pulposus Scaffold

Ethanol-Mediated Compaction and Crosslinking Enhance Mechanical Properties and Degradation Resistance While Maintaining Cytocompatibility of a Nucleus Pulposus Scaffold

Chemical imaging of a Symbiodinium sp. cell using synchrotron infrared microspectroscopy: a feasibility study

A technical review and guide to RNA fluorescence in situ hybridization

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